Abstract The fundamental organizing principle of axonal connections throughout the mammalian visual system, beginning with the projections of retinal ganglion cells (RGCs), is the arrangement of projections into retinotopic maps, organized to maintain the spatial arrangement of RGCs in the retina through the orderly terminations of their axons to the target, and thereby create a representation of the visual world in the brain critical for establishing high acuity vision. This proposal will address the molecular mechanisms that control the development of the retinotopic map within the projection of RGCs to the superior colliculus (SC), a primary target of RGCs, and the predominant model system for determining mechanisms that control the development of topographic maps in the visual system, as well as for other sensory systems. Our studies will use analyses ranging from determining the expression patterns and localization of the critical ligands, receptors and signaling molecules within RGC axons and the SC, biochemical studies to determine the receptor complexes formed by key guidance components and their influences on signaling pathways and function, use of in vitro axon guidance assays to determine the predominant function of guidance systems and receptor-ligand interactions, and in vivo analyses of the function and requirement of the guidance systems and their components using conditional knockout mice and complementary Cre lines to provide alternative and corroborative approaches. The proposed studies make use of approximately twenty lines of genetically engineered mice, and scores of compound lines. The proposal has 4 complementary Aims, each with multiple sub-Aims that test specific hypotheses suggested by our preliminary studies, previous findings, and computational modeling. Aim 1 will demonstrate a novel functional relationship between the neurotrophin receptor TrkB and the EphA family of axon guidance receptors and that the complexing of EphA and TrkB enhances the repellent activity of EphA forward signaling required for retinotopic mapping in a BDNF independent manner. Aim 2 will determine a role for BDNF - TrkB signaling in mediating the primary branching interstitially along RGC axons and branch arborization as a mechanism to complement the opposing gradients of repellent activities generated by EphA forward signaling and ephrin-A reverse signaling to generate topographic specificity in RGC axon branching and arborization. Aim 3 will directly determine for the first time the involvement of ephrin-As as receptors for the repellent activity mediated by EphAs expressed in the SC, and will define novel co-receptors required to complex with the GPI-anchored ephrin-As to mediate their reverse signaling upon binding EphAs. Aim 4 will test the prediction from computational modeling that ephrin- As present on RGC axons act as axon repellents that cooperate with ephrin-As endogenous to the SC to drive the large-scale remodeling of RGC axons through degenerative axon elimination required for the remodeling of the initially coarse retinocollicular map to establish a refined retinotopic map.